Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus that was found to have antifungal activity, particularly against Candida albicans, both in vitro and in vivo. Rapamycin is available commercially as Rapamune® (Wyeth). Rapamycin has also been shown to be useful in antitumor compositions, as an immunosuppressive agent, in the treatment of rheumatoid arthritis; in preventing or treating systemic lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonary inflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetes mellitus [Fifth Int. Conf. Inflamm. Res. Assoc. 121 (Abstract), (1990)], adult T-cell leukemia/lymphoma [European Patent Application 525,960 A1, and smooth muscle cell proliferation and intimal thickening following vascular injury [R. Morris, J. Heart Lung Transplant 11 (pt. 2): 197 (1992)].
Rapamycin and its preparation are described in U.S. Pat. No. 3,929,992, issued Dec. 30, 1975. Rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-779) is an ester of rapamycin which has demonstrated significant inhibitory effects on tumor growth in both in vitro and in vivo models. The preparation and use of hydroxyesters of rapamycin, including CCI-779, are disclosed in U.S. Pat. Nos. 5,362,718 and 6,277,983, and U.S. Patent Publication No. U.S. 2005-0033046 A1 (U.S. patent application Ser. No. 10/903,062).
Rapamycin derivatives at the 42-OH position have been synthesized and found to be useful for inducing immunosuppression, in the treatment of transplantation rejection, autoimmune diseases, solid tumors, adult T-cell leukemia/lymphoma, hyperproliferative vascular disorders, among others. Some derivatives serve as the precursors for the synthesis of rapamycin conjugates of general formula (I) below, which are useful as immunogenic molecules for the generation of antibodies specific for rapamycin as well as for isolating rapamycin binding proteins for immunoassays, and for detecting antibodies specific for rapamycin or its derivatives thereof.

In formula I, the carrier is an immunogenic carrier material or detector carrier material such as a protein or polypeptide and L is a linker, which enables the rapamycin to be attached to the carrier. [U.S. Patent Publication No. U.S. 2004-0010920].
A number of 42-derivatives of rapamycin that can be used as linking groups for the preparation of conjugates have been described. For example, the preparation of fluorinated ester of rapamycin is described in U.S. Pat. No. 5,100,883, the preparation of amide esters is described in U.S. Pat. No. 5,118,667, the preparation of aminoesters is described in U.S. Pat. No. 5,130,307, the preparation of carbamates of rapamycin is described in U.S. Pat. No. 5,118,678, the preparation of sulfonates and sulfamates are described in U.S. Pat. No. 5,177,203, the preparation of 42-ester with succinic acid and other dicarboxylic acids (adipic acid, glutaric acid, diglycolic acid, etc) are described in U.S. Patent Publication No. U.S. 2001-0010920 A1, U.S. Pat. No. 5,378,696, and International Patent Publication Nos. WO 98/45333, WO 94/25072, WO 94/25022, and WO 92/05179. In one embodiment, 42-esters with dicarboxylic acids, such as 42-hemisuccinate, 42-hemiglutarate and 42-hemiadipates (formula II) are used for the synthesis of rapamycin conjugate of formula I.

The synthesis of a compound of formula (II) has been described as being carried out by direct esterification of 42-OH with a corresponding anhydride in the presence of a weak base. Due to the sensitivity of rapamycin to basic conditions, and along with the poor regioselectivity, the desired 42-hemiester is produced with poor yield (typically below 20%) after HPLC purification; the crude product is contaminated by 31,42-diester, 31-ester and other by-products.

In an effort to improve the yield for this process, a two-step lipase-catalyzed hydrolysis approach was used [M. Adamczyk, et al, Tetrahedron Letters, 35(7):1019-1022 (1994)], in which the corresponding benzyl and methyl ester of rapamycin of 42-hemisuccinate were hydrolyzed using lipase from Pseudomonas sp. A slightly improved yield was obtained (29% from benzyl ester and 50% from the methyl ester). However, the synthesis of rapamycin 42-hemisuccinate benzyl and methyl ester via conventional chemistry also suffers from poor regioselectivity, low yield and tedious purification steps.

There is therefore a need for an efficient synthesis of hemiesters with improved yield.